Activation loop targeting strategy for design of receptor-interacting protein kinase 2 (RIPK2) inhibitors

Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pankinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed > 10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity

Structure Guided Design of Potent and Selective Ponatinib-Based Hybrid Inhibitors for RIPK1

SummaryRIPK1 and RIPK3, two closely related RIPK family members, have emerged as important regulators of pathologic cell death and inflammation. In the current work, we report that the Bcr-Abl inhibitor and anti-leukemia agent ponatinib is also a first-in-class dual inhibitor of RIPK1 and RIPK3. Ponatinib potently inhibited multiple paradigms of RIPK1- and RIPK3-dependent cell death and inflammatory tumor necrosis factor alpha (TNF-α) gene transcription. We further describe design strategies that utilize the ponatinib scaffold to develop two classes of inhibitors (CS and PN series), each with greatly improved selectivity for RIPK1. In particular, we detail the development of PN10, a highly potent and selective “hybrid” RIPK1 inhibitor, capturing the best properties of two different allosteric RIPK1 inhibitors, ponatinib and necrostatin-1. Finally, we show that RIPK1 inhibitors from both classes are powerful blockers of TNF-induced injury in vivo. Altogether, these findings outline promising candidate molecules and design approaches for targeting RIPK1- and RIPK3-driven inflammatory pathologies

Inflammatory Signaling by NOD-RIPK2 Is Inhibited by Clinically Relevant Type II Kinase Inhibitors

Summary RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers

Small molecule inhibitors reveal an indispensable scaffolding role of RIPK2 in NOD2 signaling

RIPK2 mediates inflammatory signaling by the bacteria-sensing receptors NOD1 and NOD2. Kinase inhibitors targeting RIPK2 are a proposed strategy to ameliorate NOD-mediated pathologies. Here, we reveal that RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling and show that RIPK2 inhibitors function instead by antagonizing XIAP-binding and XIAP-mediated ubiquitination of RIPK2. We map the XIAP binding site on RIPK2 to the loop between b2 and b3 of the N-lobe of the kinase, which is in close proximity to the ATP-binding pocket. Through characterization of a new series of ATP pocket-binding RIPK2 inhibitors, we identify the molecular features that determine their inhibition of both the RIPK2-XIAP interaction, and of cellular and in vivo NOD2 signaling. Our study exemplifies how targeting of the ATP-binding pocket in RIPK2 can be exploited to interfere with the RIPK2-XIAP interaction for modulation of NOD signaling

Structure-Based Design of Selective RIPK1 and RIPK2 Inhibitors

Kinases regulate various biological functions by post-translational phosphorylation of proteins. Kinase dysfunction is associated with many pathological conditions. Therefore, kinase inhibitors have become an important class of drugs and chemical biology probes for mechanistic studies of diseases. This dissertation focuses on a structural-based design of selective RIPK1 and RIPK2 inhibitors using various approaches. Chapter 1 presents an introduction to protein kinases and a review of kinase inhibitor types for understanding the rationale and design of selective RIPK1 and RIPK2 inhibitors. Chapter 2 describes two design strategies to potent and selective RIPK1 inhibition. The first strategy involves modifications of ponatinib, a type II Abl kinase inhibitor, exploiting differences in the steric and hydrophilic characteristics of Abl and RIPK1 gatekeeper residues. An introduction of tert-butyl on the central phenyl (CS5) caused unfavorable interactions with Abl’s gatekeeper and resulted in significantly improved selectivity for RIPK1 versus Abl/RIPK2/RIPK3. To improve cellular activity, a hybridization strategy linking ponatinib and Nec-1, a type III RIPK1 inhibitor, was pursued. PN10 displayed selectivity for RIPK1 inhibition and better RIPK1 cellular activity than either Nec-1 or ponatinib. In Chapter 3, strategies to develop selective type II RIPK2 inhibitors based on regorafenib, a VEGFR inhibitor, are presented. The first strategy is based on structural differences between RIPK2 and VEGFR around allosteric hydrophobic pocket and gatekeeper residues. The second strategy targeted the activation loop, a region of kinases with diverse amino acid sequences. CSR35 was identified and shown to form an interaction with the activation loop. A RIPK2CSR35 co-crystal structure revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in CSR35 and Lys169. Chapter 4 presents a hybridization strategy between ALK2 type I inhibitor LDN-214117 and B-Raf type I½ inhibitor PLX4032 as an approach to conformational distinct αC-helix-displacing RIPK2 inhibitors. Potent and selective RIPK2 inhibitors (CSLP43 and CSLP37) were achieved through modifications of the trimethoxyphenyl in LDN-214117 that occupies the hydrophobic pocket in RIPK2 next to the αC-helix. RIPK2inhibitor co-crystal structures of several derivatives suggested that they bind in a type I mode. However, further analysis of CSLP43 and CSLP37 is warranted since they display greater cellular potency.Chemistry, Department o

Characterization and structure of the human lysine-2-oxoglutarate reductase domain, a novel therapeutic target for treatment of glutaric aciduria type 1

In humans, a single enzyme 2-aminoadipic semialdehyde synthase (AASS) catalyses the initial two critical reactions in the lysine degradation pathway. This enzyme evolved to be a bifunctional enzyme with both lysine-2-oxoglutarate reductase (LOR) and saccharopine dehydrogenase domains (SDH). Moreover, AASS is a unique drug target for inborn errors of metabolism such as glutaric aciduria type 1 that arise from deficiencies downstream in the lysine degradation pathway. While work has been done to elucidate the SDH domain structurally and to develop inhibitors, neither has been done for the LOR domain. Here, we purify and characterize LOR and show that it is activated by alkylation of cysteine 414 by N-ethylmaleimide. We also provide evidence that AASS is rate-limiting upon high lysine exposure of mice. Finally, we present the crystal structure of the human LOR domain. Our combined work should enable future efforts to identify inhibitors of this novel drug target

Structure Guided Design of Potent and Selective Ponatinib-Based Hybrid Inhibitors for RIPK1

RIPK1 and RIPK3, two closely related RIPK family members, have emerged as important regulators of pathologic cell death and inflammation. In the current work, we report that the Bcr-Abl inhibitor and anti-leukemia agent ponatinib is also a first-in-class dual inhibitor of RIPK1 and RIPK3. Ponatinib potently inhibited multiple paradigms of RIPK1- and RIPK3-dependent cell death and inflammatory tumor necrosis factor alpha (TNF-α) gene transcription. We further describe design strategies that utilize the ponatinib scaffold to develop two classes of inhibitors (CS and PN series), each with greatly improved selectivity for RIPK1. In particular, we detail the development of PN10, a highly potent and selective “hybrid” RIPK1 inhibitor, capturing the best properties of two different allosteric RIPK1 inhibitors, ponatinib and necrostatin-1. Finally, we show that RIPK1 inhibitors from both classes are powerful blockers of TNF-induced injury in vivo. Altogether, these findings outline promising candidate molecules and design approaches for targeting RIPK1- and RIPK3-driven inflammatory pathologies

Synthesis, in Vitro Evaluation and Cocrystal Structure of 4‑Oxo-[1]benzopyrano[4,3‑<i>c</i>]pyrazole Cryptosporidium parvum Inosine 5′-Monophosphate Dehydrogenase (<i>Cp</i>IMPDH) Inhibitors

Cryptosporidium inosine 5′-monophosphate dehydrogenase (<i>Cp</i>IMPDH) has emerged as a therapeutic target for treating Cryptosporidium parasites because it catalyzes a critical step in guanine nucleotide biosynthesis. A 4-oxo-[1]­benzopyrano­[4,3-<i>c</i>]­pyrazole derivative was identified as a moderately potent (IC₅₀ = 1.5 μM) inhibitor of <i>Cp</i>IMPDH. We report a SAR study for this compound series resulting in <b>8k</b> (IC₅₀ = 20 ± 4 nM). In addition, an X-ray crystal structure of <i>Cp</i>IMPDH·IMP·<b>8k</b> is also presented

Inhibition and Crystal Structure of the Human DHTKD1-Thiamin Diphosphate Complex

DHTKD1 is the E1 component of the 2-oxoadipate dehydrogenase complex, an enzyme involved in the catabolism of (hydroxy-)lysine and tryptophan. Mutations in DHTKD1 have been associated with 2-aminoadipic and 2-oxoadipic aciduria, Charcot-Marie-Tooth disease type 2Q and eosinophilic esophagitis, but the pathophysiology of these clinically distinct disorders remains elusive. Here we report the identification of adipoylphosphonic acid and tenatoprazole as DHTKD1 inhibitors using targeted and high throughput screening, respectively. We furthermore elucidate the DHTKD1 crystal structure with thiamin diphosphate bound at 2.25 Å. We also report the impact of ten disease-associated missense mutations on DHTKD1. Whereas the majority of the DHTKD1 variants displayed impaired folding or reduced thermal stability in combination with absent or reduced enzyme activity, three variants showed no abnormalities. Our work provides chemical and structural tools for further understanding of the function of DHTKD1 and its role in several human pathologies